The Editor’s pick selection of the most intriguing papers is highlighted in yellow.
D-AAs AND PHYSIOLOGICAL:
Sodium benzoate attenuates 2,8-dihydroxyadenine nephropathy by inhibiting monocyte/macrophage TNF-α expression
Oshima Y., Wakino S., Kanda T., Tajima T., Itoh T., Uchiyama K., Yoshimoto K., Sasabe J., Yasui M., Itoh H.
(2023) Scientific Reports, 13 (1), art. no. 3331, DOI: 10.1038/s41598-023-30056-6
Sodium benzoate (SB) is a D-amino acid oxidase (DAAO) inhibitor which also possesses an anti-inflammatory effect. C57BL/6JJcl mice with chronic kidney disease (AdCKD) were treated with SB: it significantly attenuated AdCKD by decreasing serum creatinine and urea nitrogen levels, and kidney interstitial fibrosis and tubular atrophy scores. Furthermore, the survival of AdCKD mice improved 2.6-fold by SB. Notably SB exhibited renal protective effects in AdCKD in DAAO deficient mice, suggesting that the anti-inflammatory effect of SB was independent of the flavoenzyme activity
Characterization of E121K mutation of D-amino acid oxidase – Insights into mechanisms leading to amyotrophic lateral sclerosis
Dave U., Khan S., Gomes J.
(2023) Biochimica et Biophysica Acta – Proteins and Proteomics, 1871 (6), art. no. 140947, DOI: 10.1016/j.bbapap.2023.140947
Targeted next-generation sequencing identified the E121K mutation in D-amino acid oxidase (DAAO) as associated with amyotrophic lateral sclerosis (ALS) in patients from India. The recombinant purified E121K variant was inactive and exhibited a lower affinity for the FAD cofactor and benzoate inhibitor. Furthermore, it has higher beta-sheet content, melting temperature, and oligomeric state compared to the wild-type DAAO, and was more prone to aggregation. These results give insights into the underlying mechanisms leading to ALS pathogenesis..
Surface-Enhanced Raman Scattering Enantioselective Detection of Gastric Cancer-Related D-Amino Acids in Saliva Based on Enzyme-Mediated Cascade Reaction
Chang Liu, Jian Zhang, Qiangting Zheng, Lijun Zhao, Peng-Fei Kong, Haifeng Yang, Xinling Liu Anal Chem. 2023 Sep 5;95(35):13029-13035. doi: 10.1021/acs.analchem.3c01030. Epub 2023 Aug 23.
D-Proline (D-Pro) and D-alanine (D-Ala) are regarded as biomarkers of gastric cancer. The surface-enhanced Raman scattering (SERS) technique is efficacious for the detection of molecules but ineffective in enantiomeric differentiation. Herein, Raman-active boronate modified SERS chips are constructed to develop a D-amino acid oxidase (DAAO)-mediated cascade reaction-based SERS enantioselective assay for D-Pro and D-Ala. A linear range from 20 to 400 μmol/L and a limit of detection of 14.8 μmol/L were reached. These chips were further utilized for saliva sample analysis, and the total levels of D-Pro and D-Ala in salivary samples from gastric cancer patients were much higher than those of healthy persons
D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease
Satoko Umeda, Tomohisa Sujino, Kentaro Miyamoto, Yusuke Yoshimatsu, Yosuke Harada, Keita Nishiyama, Yoshimasa Aoto, Keika Adachi, Naoki Hayashi, Kimiko Amafuji, Nobuko Moritoki, Shinsuke Shibata, Nobuo Sasaki, Masashi Mita, Shun Tanemoto, Keiko Ono, Yohei Mikami, Jumpei Sasabe, Kaoru Takabayashi, Naoki Hosoe, Toshihiko Suzuki, Toshiro Sato, Koji Atarashi, Toshiaki Teratani, Haruhiko Ogata, Nobuhiro Nakamoto, Daisuke Shiomi, Hiroshi Ashida, Takanori Kanai
Cell Mol Gastroenterol Hepatol. 2023 Aug 9;S2352-345X(23)00148-0. doi: 10.1016/j.jcmgh.2023.08.002. Online ahead of print.
Here, the ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. D-alanine inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. The authors concluded that D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases.
Association Between Risperidone Use and Kidney Function Decline in Patients with Schizophrenia: A Retrospective Cohort Study
Megumi Oshima, Tadashi Toyama, Yusuke Nakade, Daichi Yomogida, Takahiro Yuasa, Keisuke Horikoshi, Taichirou Minami, Hisayuki Ogura, Shiori Nakagawa, Taro Miyagawa, Shinji Kitajima, Akinori Hara, Norihiko Sakai, Miho Shimizu, Masashi Mita, Masashi Kinoshita, Mitsutoshi Nakada, Mitsuru Kikuchi, Yasunori Iwata, Takashi Wada
Clin Ther. 2023 Jul 22;S0149-2918(23)00252-7. doi: 10.1016/j.clinthera.2023.07.002. Online ahead of print.
Risperidone, a used atypical antipsychotic agent for schizophrenia, is also a D-amino acid oxidase inhibitor, thus it would prevent kidney disease progression. Here, the association between risperidone use and kidney function decline in patients with schizophrenia was studied. 212 patients used risperidone and 1468 patients had no record of risperidone use. The incidence rate of 40% eGFR decline was lower in the risperidone group than in the control group. After adjustment for baseline age, sex, and eGFR, risperidone use was associated with a decreased risk for 40% eGFR decline.
D-AAs AND BACTERIA:
A design of experiments screen reveals that Clostridium novyi-NT spore germinant sensing is stereoflexible for valine and its analogs
Sundaresan A., Le Ngoc M., Wew M.U., Ramkumar V., Raninga P., Sum R., Cheong I.
(2023) Communications Biology, 6 (1), art. no. 118, DOI: 10.1038/s42003-023-04496-9
Clostridium novyi-NT is known as an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells. This study discovered that D-valine (and its analogs) is a potent germinant, inducing 50% spore germination at 4.2 mM concentration, as well as L-cysteine. On the contrary, D-alanine inhibits all germination, even in complex growth media.
D-AAs & BIOTECHNOLOGY:
Design, synthesis and evaluation of 177Lu-labeled inverso and retro-inverso A9 peptide variants targeting HER2-overexpression
Amit Kumar Sharma, Rohit Sharma, Haladhar Dev Sarma, Archana Mukherjee, Tapas Das, Drishty Satpati
Bioorg Chem. 2023 Nov;140:106761. doi: 10.1016/j.bioorg.2023.106761. Epub 2023 Aug 4.
Several HER2-specific peptides are investigated to develop an effective radiopharmaceutical for clinical screening of breast cancer patients. Here, starting from the [177Lu]DOTA-L-A9 peptide an ‘inverso’ peptide with all D-amino acids and a ‘retro-inverso’ peptide where sequence of D-amino acids was reversed were designed. The two radiopeptides [177Lu]DOTA-D-A9 and [177Lu]DOTA-rD-A9 exhibited significantly improved in vivo metabolic stability over the original L-peptide and the latter demonstrated better pharmacokinetic behavior with significantly higher tumor uptake than the inverso peptide and the original peptide.
Engineering acetylation platform for the total biosynthesis of D-amino acids
Yanqi Bi, Jingyu Wang, Jialong Li, Hsiang-Hui Chou, Tianhua Ren, Jinlin Li, Kechun Zhang
Metab Eng. 2023 Sep 9;80:25-32. doi: 10.1016/j.ymben.2023.09.001. Online ahead of print.
Here, an artificial metabolic pathway was designed by engineering bacteria to heterologously express racemase and N-acetyltransferase to produce N-acetyl-D-amino acids from L-amino acids, thus avoiding the cytotoxicity of D-amino acids. A total of sixteen amino acid targets have been evaluated. By pathway optimization and metabolic engineering in Escherichia coli N-acetyl-D-valine, N-acetyl-D-serine, N-acetyl-D-phenylalanine and N-acetyl-D-phenylglycine were produced at gram/liter scale.
ENZYMES ACTIVE ON D-AAs:
Directed evolution of Rhodotorula gracilis d-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening
Christoph Küng, Rosario Vanella, Michael A Nash
React Chem Eng. 2023 Apr 20;8(8):1960-1968. doi: 10.1039/d3re00002h. eCollection 2023 Jul 25.
In this work a high-throughput screening and a directed evolution strategy relying on single-cell hydrogel encapsulation was developed to enhance the performance of D-Amino acid oxidase from Rhodotorula gracilis (RgDAAO). By screening a RgDAAO variant library containing ∼106 clones while lowering the D-Ala substrate concentration, four variants displayed on the yeast surface were identified, showing a 5-fold lower Km than the parent enzyme. The substitutions were scattered across the RgDAAO structure, demonstrating the difficulty in rationally predicting allosteric sites.
Novel tetrahydrofolate-dependent D-serine dehydratase activity of serine hydroxymethyltransferases
Tetsuya Miyamoto, Shinya Fushinobu, Yasuaki Saitoh, Masae Sekine, Masumi Katane, Kumiko Sakai-Kato, Hiroshi Homma
FEBS J. 2023 Sep 12. doi: 10.1111/febs.16953. Online ahead of print.
Here, a novel D-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) was identified in bacteria as well as in mammals. SHMT is known to catalyze the conversion of L-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, both human and Escherichia coli SHMTs have D-serine dehydratase activity, which degrades D-serine to pyruvate and ammonia. SHMT required THF to catalyze D-serine dehydration; did not exhibit dehydratase activity toward the L-enantiomer; did not use D-serine as a substrate in the canonical hydroxymethyltransferase reaction. The D-serine dehydratase activity of human SHMT was inhibited by high concentration of THF, whereas that of E. coli SHMT was increased. The catalytic efficiency of dehydratase activity was lower than that of hydroxymethyltransferase activity. The D-serine dehydratase activity of SHMT is of main physiological relevance because D-serine inhibited the growth of an SHMT deletion mutant of E. coli more than that of the wild-type strain. SHMT is thus involved in both L- and D-serine metabolism.
Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering
Liuyu Wang, Heng Tang, Hongli Zhu, Yaping Xue, Yuguo Zheng
Biotechnol Bioeng. 2023 Aug 31. doi: 10.1002/bit.28541. Online ahead of print.
D-Amino acid oxidase (DAAO) can be used for synthesizing optically pure L-amino acids, including L-phosphinothricin ( L-PPT), a chiral herbicide. However, the native DAAOs have low activity against the unnatural substrate D-PPT. The kinetic properties of DAAO from Rhodotorula taiwanensis (RtwDAAO) toward D-PPT was increased through protein engineering mainly based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M3rd -SHVG exhibited a >2000-fold increase in relative activity. Molecular dynamics simulation revealed that the M3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme-substrate binding, allowing efficiently catalyze the reaction on unnatural substrates. By using this variant, 500 mM D,L-PPT was completely converted.
Development of a novel ultrasound- and biocrosslinking-enhanced immobilization strategy with application to food enzymes
Han J., Zhang T., Zhou Z., Zhang H.
(2023) Food Chemistry, 417, art. no. 135810, DOI: 10.1016/j.foodchem.2023.135810
This work focuses on a novel immobilization method based on the tyrosine-tag crosslinking mechanism using food enzymes such as D-amino acid oxidase and glucose dehydrogenase (immobilized on macroporous resins, amino resins, epoxy resins, and multiwalled carbon nanotubes). D-amino acid oxidase was immobilized on macroporous resins with an increased yield by 24%, enhanced reusability, and higher sensitivity when an enzyme electrochemical sensor was constructed.
In search for structural targets for engineering D-amino acid transaminase: modulation of pH optimum and substrate specificity
Shilova S.A., Matyuta I.O., Khrenova M.G., Nikolaeva A.Y., Klyachko N.L., Minyaev M.E., Khomutov A.R., Boyko K.M., Popov V.O., Bezsudnova E.Y.
(2023) The Biochemical journal, 480 (16), pp. 1267 – 1284, DOI: 10.1042/BCJ20230233
This work focuses on the D-amino acid transaminase from Aminobacterium colombiense. Site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis allowed to identify the active site residues responsible for substrate binding and selectivity, thermostability of a functional dimer, and pH optimum. The authors demonstrated that the high specificity toward D-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue; the K237A substitution shifts the catalytic activity optimum to acidic pH.
Characteristics of alanine racemase in Lactobacillus sakei ZH-2 strain
Kanauchi M., Matsumoto N.
(2023) Food Science and Nutrition, 11 (8), pp. 4745 – 4755, DOI: 10.1002/fsn3.3452
Some D-amino acid functions for food production are widely known: the enzyme alanine racemase (ALRase) in Lactobacillus sakei ZH-2 was analyzed in this study. Complete genome sequencing revealed that the amino acid sequence of ALRase in ZH-2 strain has a 99.4% similarity with the same protein from Lactobacillus sakei 23K strain. ALRase from ZH-2 strain has approximately 38.0 kDa molecular mass (by SDS-PAGE), an optimum at pH 9.0 at 30–40°C, and a stability at pH 9.0–10.0 and 4–40 °C. Its cofactor is pyridoxal phosphate and the activity is stimulated by copper and zinc ions. ALRase best reacted with alanine while a 40% activity was measured on serine.
D-AAs AND ANALYTICAL METHODS:
D-Amino acid recognition of tripeptides studied by ultraviolet photodissociation spectroscopy of hydrogen-bonded clusters
Inoue K., Fujihara A.
(2023) Amino Acids, 55 (7), pp. 931 – 938, DOI: 10.1007/s00726-023-03284-3
The D-amino acid recognition of peptides was investigated here using a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. The UV photodissociation spectroscopy and water adsorption of hydrogen-bonded protonated clusters of tryptophan enantiomers and tripeptides (SAA, ASA, and AAS) were studied at 8 K in the gas phase. This study showed that the indole ring of Trp was located on the surface of H+(D-Trp)ASA, and the amino and carboxyl groups of Trp formed hydrogen bonds in H+(D-Trp)ASA. For the other five clusters, the indole rings of Trp were hydrogen bonded in the clusters, and the amino and carboxyl groups of Trp were present on the cluster surfaces.
Two-dimensional LC-MS/MS and three-dimensional LC analysis of chiral amino acids and related compounds in real-world matrices
Ishii C., Hamase K.
(2023) Journal of Pharmaceutical and Biomedical Analysis, 235, art. no. 115627, DOI: 10.1016/j.jpba.2023.115627
This review focuses on multi-dimensional HPLC methods for the determination of chiral amino acids, especially two-dimensional LC-MS/MS and three-dimensional LC methods, and their applications to a variety of real-world matrices are summarized.
Separation and Identification of Isoleucine Enantiomers and Diastereomers Using an Original Chiral Resolution Labeling Reagent
Makoto Ozaki, Motoshi Shimotsuma, Takefumi Kuranaga, Hideaki Kakeya, Tsunehisa Hirose
Chem Pharm Bull (Tokyo). 2023 Aug 24. doi: 10.1248/cpb.c23-00439. Online ahead of print.
Among the 20 amino acids that make up proteins, threonine (Thr) and isoleucine (Ile) have two chiral carbons and thus have two enantiomers and diastereomers. The separation and identification of these stereoisomers by HPLC are laborious and complicated. Here, an analytical method for the separation and identification of Ile stereoisomers through liquid chromatography-mass spectrometry based on a chiral resolution labeling reagent (1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine-amide, L-FDVDA) and a PBr column packed with pentabromobenzyl-modified silica gel was set up. Twenty D,L-amino acids including Thr stereoisomers (41 amino acids including glycine) were separated and identified using C18 column, while Ile stereoisomers were separated using a PBr column. Additionally, peptides containing Thr and Ile stereoisomers can be accurately detected through labeling with L-FDVDA.
The D-amino acids International Research Center “DAAIR“ has been established in Gerenzano (Varese, Italy) in 2019 with the aim to support and perform scientific research projects and activities on the field of D-amino acids. The Center, located inside the Fondazione Istituto Insubrico Ricerca per la Vita, is aimed to represent a pole of excellence at international level for dissemination and research involving the D-amino acids (Director Silvia Sacchi).
The guiding principle is support the research projects aimed to investigate the involvement of D-amino acids in main physiological processes, from bacteria to humans. The ultimate goal is to actively participate to the elucidation of the mechanisms by which the D-amino acids perform specific functions, and to identify their presence and concentration in different organisms and compartments, also with regards to well-established functional states, with particular emphasis to pathological states. Understand the involvement of D-amino acids in important diseases as a way to set up novel therapeutic strategies.
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